Autophosphorylation of the Intracellular Domain of the Epidermal Growth Factor Receptor Results in Different Effects on Its Tyrosine Kinase Activity with Various Peptide Substrates

The effect of autophosphorylation on the tyrosine kinase activity of the epidermal growth factor receptor (EGFR) is not well understood. We previously demonstrated that phospholipase C-y physically associates with the EGF-activated EGFR, but not with a kinasenegative mutant of the EGFR, and, moreover, that only the tyrosine-phosphorylated EGFR is able to associate with phospholipase C-y. We have now investigated the effect of autophosphorylation on the tyrosine kinase activity of the EGFR by employing the purified kinaseactive intracellular domain of the EGFR (EGFR-IC) produced by a baculovirus expression system. Synthetic peptides, including ones which contain the individual major tyrosine phosphorylation sites of phospholipase C-y, were used as substrates. We found that the extensively prephosphorylated EGFR-IC exhibited similar eaction kinetics to the unphosphorylated EGFR-IC when angiotensin I1 was used as a nonspecific substrate. In contrast there was a clear stimulation of kinase activity due to autophosphorylation of the EGFR-IC when peptides representing either the major autophosphorylation site of the EGFR or the EGFR phosphorylation sites of phospholipase C-y were used as substrates. However, the modes of stimulation for these peptides differed. The binding affinity (K,) for the unphosphorylated EGFR-IC for the peptide containing Tyr-771 of phospholipase C-y was relatively poor compared with other peptides, but improved 56-fold when the EGFR-IC was prephosphorylated. On the other hand, autophosphorylation improved the r action velocity (Vm) of the phosphorylation of other peptides by 2-3-fold, with little or no increase in affinity. These results suggest that autophosphorylation of the EGFR may induce a conformational change of its kinase domain which enhances its kinase activity with exogenous substrates and may induce association with phospholipase Cy by increasing its affinity to a d main containing Tyr-771.